Studies were initiated to evaluated the role of glutamine as a respiratory fuel and to determine the fate of its carbon and nitrogen atoms for intermediary metabolism necessary for tumor growth. To facilitate this investigation, automated GC/MS methods were developed to simultaneously quantify and measure the isotopic content in amino acids, various sugars, lipids, cholesterol, and TCA intermediates. Preliminary in vitro studies with L1210 ascites tumors grown in mice were conducted to develop the approach to be used for in vitro studies with human tumor lines and in tumors and normal tissue in vivo. Incubation of L1210 tumors with various isotopic variants of glutamine and uniformly labeled glucose resulted in the following observations. (1) Large amounts of glucose and glutamine were consumed averaging 7.5 Mumoles/min-g dry weight and 2.2 Mumoles/min-g dry weight, respectively. (2) 80%-90% of (1,2,3,4,5,6-13C) glucose used was metabolised to (1,2,3-13C) lactate. (3) 89% of the 5-amido nitrogen was recovered as (15N) H3 and 80% of the alpha nitrogen underwent transamination to form (15N) alanine. Small amounts of the glutamine alpha 15N were recovered in glutamate, aspartate, proline, serine, and pyroglutamate. (4) Preliminary data indicate that glutamine transaminase activity may be present in L1210 tumors resulting in an alternate pathway to alpha-ketoglutarate via transmination to alpha- ketoglutaramate and omega amidase hydrolysis. (5) Incubation of L1210 tumors with (2, 3, 3, 4, 4-(2H)-glutamine provided evidence of a truncated TCA cycle, with glutamine supplying most of the carbons for the TCA cycle and cytosolic pyruvate supplying minimal acetylCoA for oxidation in the TCA cycle. Most of the citrate formed in the mitochondria was probably exported to the cytosol.